Cell membrane permeability
Cell membrane permeability
(Guided Inquiry topic)
Required Readings: Russell et al. (2015), (Chapter 5)pg 97-119.(review as necessary)
– Appendix F: Micropipettes
- Appendix H: Using a spectrophotometer
- Your “One stop shop to an A on your GI manuscript” document
- http://www2.mtroyal.ca/~tnickle/stylesguide (for demonstrations of how to display data).
Key Terms: Beetroot (Beta vulgaris) Cell wall, cell membrane, cytoplasm, tonoplast, central
vacuole, betacyanin. Cork borer, beet puck, blank, negative control, treatment tube, extraction tube.
Objectives
- Use an experiment to test the “fluid mosaic” model of membrane structure.
- To investigate, using proper experimentation, some of the influencesupon membrane integrity in a plant system.
- review how to graph data using Excel
- modify this labs protocol/study system to investigate your own hypothesis
INTRODUCTION
Cell membranes function to separate the contents of the cell from its external environment and to organize the cellular compartments (this applies to eukaryotes only; remember prokaryotes typically only have a plasma membrane). Membranes allow the passage of certain molecules and ions into and out of the cell and partition them within appropriate intracellular compartments.
In general, the lipid bilayer of a biological membrane is impermeable to most polar molecules because of its hydrophobic interior. This prevents the water-soluble contents of the cell from escaping. Synthetic protein-free lipid bilayers have been used to study the permeability of membranes. In vitro experiments show that almost any molecule will diffuse across these bilayers given enough time. The rate of diffusion depends upon the size of the molecule and its relative solubility in oil. Molecules that are smaller and more hydrophobic or nonpolar dissolve more rapidly across them. Large, hydrophilic molecules move diffuse across more slowly.
Study system
In this exercise you will use living beet cells to relate the structure of a membrane to its function. Beet cells contain a red pigment called betacyanin. This pigment is located in the large central vacuole of the cell, which is located entirely within the cytoplasm of plant cells and surrounded by a membrane called the tonoplast (tonoplast is the proper term for the membrane surrounding the central vacuole). The cell itself is surrounded by another phospholipid bilayer, the plasma membrane. As long as both these membranes remain intact, the betacyanin will remain within the cell. If the membrane permeability is increased, however, the betacyanin will leak out and cause the fluid around the cells to become red (Figure 1).
In this exercise, you will subject discs of beet root to temperature stresses and assess the amount of damage to the cells by measuring the leakage of betacyanin into the environment.The amount of betacyanin leaked from the cell can be easily measured using a spectrophotometer. Betacyanin absorbs light maximally at 525 nm, and so as more betacyanin is leaked from the cell, there is an increase in absorbance (also known as the optical density – OD). You will be measuring the OD525 using a spectrophotometer which will be demonstrated by your instructor. The instructions for this piece of equipment can be found in Appendix H if you need to review.
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- b. *note the striations c.
Figure 1. Visualization of the location of betacyanin within the tonoplast of the beet (a), cross-section of beet to show striations in betacyanin content (b), and (c) shows our study system to manipulate.
Exercise – Procedure for temperature treatment experiment using beet tissue
Open the Adobespark video. It will run through the steps of the experiment written below. You can begin with reading muted text steps below, then watching them carried out in the video, pausing between steps if needed. You will have to watch the video to collect resulting data for this experiment.https://spark.adobe.com/video/0CyuH9QJzzhFv
***You will be graphing these results and submitting them at the end of the lab.
- Select a large, fresh beet root located on side bench or in the fridge.
- Using a cork borer, obtain a long cylinder of beet root. Use a razor blade and a ruler to cut roughly 8 disks that are 5 mm long (pay attention to the colour of your pieces as some will be lighter and some will be darker due to the naturally occurring striations of betacyanin. Try to choose one or the other for all your disks). This will give you more than enough disks for the experiment. Cut the epidermis away at each end of the cylinder and avoid including any tissue within 5 mm of the epidermis.
- Rinse all disks in a beaker of cool tap water for about 10 minutes to remove pigment from damaged cells. The disks may be left in a small amount of water until you are ready to use them. While they are rinsing carry on with step 4 below paying attention to **highlighted note**.
- Obtain 11 test tubes; 5 will be for the 1 minutetreatment, 5 will be for the 30 minuteextraction, and 1 will be for a ‘blank’ to zero your spectrophotometer. Label 5 of the test tubes for the temperature treatments (0, 20, 40, 60, and 80ºC) and fill each of these test tubes with 5 ml of water. **Immediately place these tubes into their corresponding temperature treatment baths and carry on preparing your beet tissue.** Label the corresponding extraction tubes with the same temperatures and fill with 5 ml of water.
**Your temperature experiment is written out on the next page in the form of a flow diagram to help clarify the procedure (Figure 2).**
-“Stab” the beet with cork-borer Slice with razor blade in to 5 mm pieces (‘pucks’)
(pull out ‘core’ of beet tissue)
-(Rinse pucks in tap water for ~10 minutes)
-PLACE TUBES CONTAINING DISTILLED WATER IN EACH OF 5 TEMPERATURETREATMENT WATERBATHSTO REACH INDICATED TEMPERATURE
When tubes are at appropriate temperature, Place a ‘puck’ into each of 5 test tubes containing 5 mls
distilled water.TREAT FOR ONE MINUTE.
-POUR OFF WATER (tip pucks on to separate pieces of kimwipe or a tea
sieve) (keeping your treatment ‘pucks’ separate)!
Gently, place each puck into clean test tubes of new clean water all at room temperature and extract for 30 minutes.
After 30 minutes, agitate one more time and once the puck has settled, place tubes in the spec and record resulting absorbance readings (after zeroing the spec with water)).
Figure 2. Flow diagram of a membrane permeability experiment using beet tissue (Beta vulgaris) subjected to increasing temperature treatments, followed by extraction of betacyanin in tap water quantified via a spectrophotometer measuring absorbance (of light).
(Once the water in the tubes is at the same temperature as the water bath):
- Take one of the beet disks and immerse it into the treatment tube you labeled as 0ºC that is in the ice water bath and let it sit for exactly one minute.
- After one minute, carefully remove the disk by draining the water into the sink and tapping the disk onto a kimwipe or into a small sieve. Immediately place it into the extraction tube of water labeled 0ºC. Leave the tube at room temperature for 30 minutes (in the room temperature bath) to allow extraction of the betacyanin from the disk in response to having been exposed to 0 ºC. Record the exact time you start the extraction to ensure you are precise with when you read the absorbance.
- Repeat steps 2-7 for the remaining temperature treatments. Once all 30 minute temperature response extractions have begun, turn the spectrophotometer on and set the wavelength to 525 nm.
- During the extraction period, agitate the beet root disk by gently vortexing tubes every 10 minutes.
- After 30 min, vortex the tube one last time. Blank your spectrophotometer first with your test tube of water then place each tube of betacyanin extract into the spectrophotometer and record the absorbance. You may want to blank repeatedly between absorbance readings to prevent ‘drift’.
- Once all the data is recorded beet disks can be discarded in the garbage.
- Collect results from other groups in the class. Determine the average absorbance at each temperature and standard deviation. (Report the actual temperatures your baths are at).
Table 1. Absorbance caused by betacyanin leakage from beet tissue samples in response to 1 minute temperature treatment.
| Absorbance readings at each temperature | |||||
| Trials | 0ºC | 20ºC | 40ºC | 60ºC | 80ºC |
| 1 | |||||
| 2 | |||||
| 3 | |||||
| Average | |||||
| Standard deviation | |||||
- Graph your results complete with concise and all-inclusive figure heading (graph paper provided on the next page).
Figure.________________________________________________________________ _________________________________________________________________________________
Study Questions
- Why did you remove the epidermis of the beet sample?
- Why do you put your room temperature tubes in a water bath?
- You were very careful to calibrate your spectrophotometers, measure volumes, etc. yet you likely noticed in some cases, large variation in absorbance readings within treatments. What are some reasons for this variation? Are there other changes to methodology you might include?
**During your break-out session think of another variable you and your partner/group can study that might affect membrane permeability and what affect you think it might have. Use the “One stop shop …” document for help.
** SEE LAST PAGE FOR GROUP WORKSHEET **
ALSO….
Here is an example of a modified experimental protocol using detergent in this instance (rather than temperature.
Your groups worksheet:
Group member names (in full):
Which variable do you choose? ___________________________________
What is your working hypothesis? This must be stated in an “if, …then…” statement.
This will ensure you provide a prediction rather than a question. Thinking about the structure of a membrane, what aspects of it do you think the variable of (x) would affect? As you increased the concentration of (x) do you predict the effect to increase, decrease, stay the same? If you chose pH, do you predict a linear response to increasing/decreasing pH?
________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
Any additional comments to add to your rationale? ___________________________________ __________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
